MITE insertions generate enhancers for iron transporter CwIRT1 activation in citrus

Abstract Iron (Fe) deficiency constrains citrus productivity in alkaline soils. Although the high-affinity Fe transporter IRT1 is essential for Fe acquisition, its transcriptional regulation in citrus remains unclear. Here, we reveal that differential tolerance to Fe deficiency between Fe-sensitive Poncirus trifoliata (P. trifoliata) and Fe-tolerant Citrus wilsonii Tanaka (C. wilsonii) is linked to rapid induction of CwIRT1. We further identify two unique miniature inverted-repeat transposable elements (MITE1 and MITE2) in the promoter region of CwIRT1, which act as Fe-deficiency-responsive enhancers. These two MITEs respectively generate binding sites for the transcription factors CwERF12 and CwARF12, both of which are upregulated under low-Fe conditions. CRISPR/Cas9-mediated dual knockout of CwERF12 and CwARF12, but not single mutations, suppresses CwIRT1 expression and reduces root Fe uptake. We propose that MITEs carrying TF-binding motifs can serve as enhancers recruiting stress-responsive transcription factors to activate CwIRT1, highlighting transposable element-mediated cis-regulatory as a driver of nutrient stress adaptation in perennial crops.